Effects Of Gen On The Migration And Invasion Of The RA-FLS Through P38 Pathway

ObjectiveRheumatoid arthritis (RA) is a chronic systemic autoimmune disease, and synovitis is the basic pathological change of RA. Fibroblast-like synoviocytes (FLS) located in synovial lining layer of RA patients has similar characteristics to tumor cells in adhesion to ECM, migration and invasion, therefore, FLS played a central role in pathogenesis of RA.Our previous studies have shown genistein (Gen) could inhibit the proliferation of FLS from CIA rats and the secretion of TNF-α and IL-1β, and reduce the joint inflammation and inhibit the joint destruction in CIA rats. Evdences have also shown that Gen could suppress tumor cells adhesion to ECM, migration and invasion. Therefore, this study designed to explore effects of Gen on adhesion to ECM, migration and invasion of RA-FLS.Methods1. Human RA-FLS strain (MH7A) was used to examine effects of Gen on cell viability in RA-FLS, and concentrations of Gen which had no significant influence on cell viability was selected for the following research.2. Cell-matrix adhesion assays were used to examine effects of Gen on adhesion to different types of ECM (Collagen Ⅰ, Fibronectin, Matrigel) of RA-FLS.3. Cell wound-healing assays and transwell cell migration assays were used to examine effects of Gen on migration in RA-FLS.4. Transwell cell invasion assays were used to examine effects of Gen on invasion in RA-FLS.5. The immunofluorescence assays were applied to observe the effects of Gen on the actin cytoskeleton rearrangement in RA-FLS, such as the formation of filopodia and lamellipodia.6. Western blot assays were used to detect the expression of ERK, p-ERK, JNK, p-JNK, p38, p-p38 and MMP-2.Results1. The results of CCK-8 assays showed that rates of cell viability in RA-FLS incubated with Gen at concentrations of 20 μM,40 μM and 60 μM for 24 h were 98.30±2.90%, 95.70±5.65%,92.70±7.01% compared with the normal control group; and there were no significant effect of Gen on cell viability of RA-FLS (p>0.05). Thus,20 μM,40 μM and 60 μM of Gen were used in following research.2. Results from cell-matrix adhesion assays showed that adhesive ratios of RA-FLS adhesion to Collagen I at concentrations of 20 μM,40 μM and 60μM were 97.43±1.18%, 88.92±4.23% and 78.25±4.90%, adhesive ratios of RA-FLS adhesion to Fibronectin were 95.18±2.00%,89.35±2.35% and 78.43±3.27%, adhesive ratios of RA-FLS adhesion to Matrigel were 96.35±2.13%,84.96±3.21% and 75.82±1.38%, respectively. Statistical analysis demonstrated that Gen inhibited cell-matrix adhension of RA-FLS in 40 μM group and 60 uM group (p<0.05), especially in 60 μM group (p<0.05), no significant effect was shown in 20 μM group (p>0.05).3. Results from cell wound-healing assays demonstrated that Gen inhibited the ability of migration in RA-FLS at concentrations of 20 μM,40 μM and 60 μM; data from transwell cell migration assays demonstrated that migratory ratios of RA-FLS with Gen were 97.10±5.16%,77.97±5.44% and 51.95±5.26% at concentrations of 20 μM,40μM and 60 μM. Statistical analysis indicated that Gen inhibited migration of RA-FLS in 40μM group and 60 μM group (p<0.05), especially in 60μM group, and no significant effect was shown in 20 μM group (p>0.05).4. Results from transwell cell invasion assay demonstrated that invasive ratios of RA-FLS with Gen were 94.79±5.24%,64.54±10.13% and 51.32±12.36% at concentrations of 20 μM,40 μM and 60 μM. Statistical analysis demonstrated that Gen inhibited invasion of RA-FLS in 40 μM group and 60 μM group (p<0.05), especially in 60 μM group, and no significant effect was shown in 20 μM group (p>0.05).5. Results from the immunofluorescence assay demonstrated that Gen decreased the formation of filopodia and lamellipodia in RA-FLS, obviously in 60 μM group.6. Results of the Western blot assays demonstrated that Gen downregulated expression levels of p-p38 at concentrations of 40μM and 60 μM (p<0.05), but had no significant effect on the expression levels of p-JNK, JNK, p-ERK, ERK and MMP-2 (p>0.05).Conclusions1. Gen inhibited the adhesion to ECM, migration and invasion of RA-FLS at concentrations of 40 μM and 60 μM, but had no significant effect at the concentration of 20 μM.2. Our results demonstrat that Gen may inhibit RA-FLS adhesion to ECM, migration and invasion possibly mediated by the F-actin cytoskeleton rearrangemen, such as decreasing the formation of filopodia and lamellipodia, via p38 pathway.